KR20100023538A - Manufacturing method of solid state reagent and microfluidic device accommodating the reagebt therein - Google Patents

Abstract

PURPOSE: A method for manufacturing a solid reagent and a microfluidic device receiving the solid reagent are provided to manufacture a frozen and dried solid reagent by eliminating moisture from the frozen reagent after injecting a liquid reagent into a receiving part and freezing the reagent and to receive the frozen and dried solid reagent. CONSTITUTION: A method for manufacturing a solid reagent comprises: a step of preparing a mold in which a plurality of reagent receiving parts(301) are included; a step of injecting a liquid reagent into a plurality of the reagent receiving parts; a step of freezing the liquid reagent; a step of separating the frozen reagent from the mold; and a step of eliminating a moisture from the frozen reagent and drying the reagent. The step for drying the reagent includes a step of sublimating the moisture which is contained in the frozen reagent.

Description

Manufacturing method of solid state reagent and microfluidic device accommodating the reagebt therein

A method of preparing a solid phase reagent from a liquid phase reagent and a microfluidic device containing the solid phase reagent are disclosed.

Various methods of analyzing samples have been developed in various application fields such as environmental monitoring, food inspection, and medical diagnostics. However, existing inspection methods require a lot of manual work and various equipment. In order to perform the test according to a predetermined protocol, a skilled experimenter must manually perform various steps such as injecting, mixing, separating and moving reagents, reaction, and centrifugation several times. It causes the error.

Skilled clinical pathologists are needed to perform the test quickly. Even experienced clinical pathologists have a lot of difficulties in performing several tests simultaneously. However, in the diagnosis of emergency patients, quick test results are necessary for quick first aid. Therefore, there is a need for an apparatus capable of simultaneously and quickly and accurately performing various pathological examinations necessary for a situation.

In the case of conventional pathological examination, large and expensive automation equipment is used, and a relatively large amount of test substance such as blood is required. It takes a long time to collect the test substance from the patient, and then receive the results in as little as 2-3 days or as long as 1-2 weeks.

To remedy this problem, miniaturized and automated equipment has been developed that can rapidly analyze test materials taken from one or a few patients as needed. As an example, when blood is injected into a disk-type microfluidic device and the microfluidic device is rotated, serum separation occurs by centrifugal force. The separated serum is mixed with a certain amount of diluent and transferred to a number of reaction chambers in the disk-like microfluidic device as well. In the plurality of reaction chambers, different reagents are pre-injected for each blood test item, and react with the serum to give a predetermined color. Blood analysis can be performed by detecting this color change.

The problem is that keeping the reagents in the liquid state is not necessarily easy. US 5,776,563 describes a method for storing lyophilized reagents and inserting them into the reaction chamber in a disk-type microfluidic device in the required amount when blood analysis is required.

Provided are a method for preparing a solid reagent from a liquid reagent and a microfluidic device containing the solid reagent.

A solid phase reagent manufacturing method according to an embodiment of the present invention, preparing a mold having a plurality of reagent receiving portion; Injecting a liquid reagent into the plurality of reagent receivers; Freezing the liquid reagent; Separating the frozen reagent from the mold; It includes; drying by removing the moisture of the frozen reagent.

As an embodiment, the drying may include subliming water contained in the frozen reagent.

In one embodiment, the liquid reagent may be concentrated to a concentration above the concentration used for the test and injected into the plurality of reagent receiving portions.

In one embodiment, at least two or more types of liquid reagents may be accommodated in the plurality of reagent accommodation portions.

In one embodiment, the shape of the plurality of accommodation portion may be at least two kinds.

In one embodiment, the mold may be a flexible material.

In one embodiment, the reagent is serum, aspartate aminotransferase (AST), Albumin (ALB), Alanine Phosphatase (ALP), alanine aminotransferase (ALT), AMY (Amylase), BUN (Urea Nitrogen), Ca ++ ( Calcium, CHOL (Total Cholesterol), CK (Creatin Kinase), Cl- (Chloide), CREA (Creatinine), D-BIL (Direct Bilirubin), GGT (Gamma Glutamyl Transferase), GLU (Glucose), HDL (High- Density Lipoprotein cholesterol, K + (Potassium), LDH (Lactate Dehydrogenase), LDL (Low-Density Lipoprotein cholesterol), Mg (Magnesium), PHOS (Phosphorus), Na + (Sodium), TCO2 (Total Carbon Dioxide), T-BIL (Total Bilirubin), TP (Total Protein), TRIG (Triglycerides), UA (Uric Acid), ALB (Albumin), TP (Total Protein) can be selected from the reagents for testing.

In one embodiment, a filler may be added to the liquid reagent. In one embodiment, the filler may be bovine serum albumin (BSA), polyethylene glycol (PEG), dextran, mannitol, polyalcohol, myo-inositol, citric Acid (citric acid), EDTA2Na (ethylene diamine tetra acetic acid disodium salt), BRIJ-35 (polyoxyethylene glycol dodecyl ether) can be selected.

In one embodiment, a surfactant may be added to the liquid reagent. In one embodiment, the surfactant is a polyethylene (polyoxyethylene), lauryl ether (lauryl ether), octooxynol (octoxynol), polyethylene alkyl alcohol, nonylphenol polyethylene glycol ether; Ethylene oxide (ethylene oxid), ethoxylated tridecyl alcohol, polyoxyethylene nonylphenyl ether phosphate sodium salt, sodium dodecyl sulfate sulfate).

Microfluidic device according to an embodiment of the present invention, the sample chamber is accommodated the sample to be inspected; A dilution chamber in which a diluent is accommodated; A reagent chamber containing a solid phase reagent; And a channel connecting the chambers.

In one embodiment, the microfluidic device may further include a valve for controlling the flow of the fluid through the channel. In an embodiment, the valve may use a material opened by electromagnetic energy as a valve material. In one embodiment, the valve material may be selected from a phase change material or a thermoplastic resin whose phase is changed by energy. In one embodiment, the valve material may include fine heating particles dispersed in the phase change material and absorbing energy of electromagnetic waves to generate heat.

Microfluidic device according to an embodiment of the present invention, the substrate is provided with a plurality of chambers; And a solid phase reagent contained in at least one of the plurality of chambers, wherein the solid phase reagent may be a quantitative reagent.

In one embodiment, the microfluidic device includes a sample chamber in which a sample to be inspected is accommodated; A dilution chamber in which a diluent is accommodated; And a plurality of reagent chambers in which the solid phase reagent is accommodated.

In one embodiment, two or more types of the solid reagent are accommodated in the plurality of reagent chambers, and the solid reagent may have a different shape according to its type.

In one embodiment, the microfluidic device includes a channel connecting the plurality of chambers; And a valve installed in the channel to control the flow of the fluid through the channel, the valve blocking the channel in the solidified state and melting by electromagnetic wave energy to open the channel.

As described above, according to embodiments of the present invention, a lyophilized solid phase reagent may be prepared, and a microfluidic device containing the lyophilized solid phase reagent may be provided.

 Hereinafter, with reference to the accompanying drawings will be described embodiments of the present invention;

1 shows a first embodiment of a microfluidic device according to the present invention. 2 and 3 are cross-sectional views of the first embodiment of the microfluidic device 100 shown in FIG. 1 and 2, the platform 1 is provided with a microfluidic structure for providing a space in which the fluid can be accommodated and a flow path through which the fluid can flow. The platform 1 may be made of plastic material such as acrylic, PMMA, COC, etc., which is easy to mold and whose surface is biologically inert. However, the material of the platform 1 is not limited thereto, and any material having chemical, biological stability, optical transparency, and mechanical processability is sufficient. As shown in FIG. 2, the platform 1 may have a two-plate structure including a lower plate 11 and an upper plate 12. In addition, as shown in FIG. 3, the platform 1 is provided with a partition plate 13 for defining a space in which the fluid can be received and a flow path through which the fluid can flow between the lower plate 11 and the upper plate 12. It may be a structure. The lower plate 11, the upper plate 12, the partition plate 13 may be coupled to each other by various methods such as bonding using a double-sided adhesive tape or adhesive, ultrasonic welding. In addition, the platform 1 may have various forms.

A series of structures for the blood test disposed in the platform 1 will be described in more detail. The platform 1 is provided with a sample chamber 10. The sample chamber 10 is for accommodating a sample, for example, blood, serum, and the like. The dilution chamber 20 is for receiving a diluent for diluting the sample to the concentration necessary for the test. The diluent can be, for example, a buffer solution or DI (distilled water). The reagent chamber 30 contains a reagent for detecting whether a sample contains a specific substance.

The sample chamber 10 is connected with the dilution chamber 20. The dilution chamber 20 is connected with the reagent chamber 30. Valves 51 and 52 are positioned between the sample chamber 10 and the dilution chamber 20 and between the dilution chamber 20 and the reagent chamber 30, respectively, to control the flow of fluid between the chambers. Although not shown in the drawing, the platform 1 may be provided with inlets for injecting a sample, a diluent, a reagent, and the like, and an air vent for discharging the air therein.

The reagent chamber 30 is irradiated with light in a sample analysis process to be described later, and at least a region where the reagent chamber 30 of the platform 1 is located is formed of a material capable of passing light.

Various types of microfluidic valves may be employed as the valves 51 and 52. The valve may be a valve opened and closed by the moving speed of the fluid in the microfluidic structure. In other words, it may be a valve that is manually opened when a predetermined pressure or more is applied by the moving speed of the fluid. For example, there are capillary valves using a microchannel structure, siphon valves, hydrophobic valves having a hydrophobic surface treatment, and the like. In this case, the valve can be controlled by the rotational speed of the microfluidic device. That is, when the rotation speed of the microfluidic device is increased, the pressure applied to the fluid in the microfluidic structure is increased. When the pressure is higher than a predetermined pressure, the valve is opened to allow the fluid to flow.

In addition, the valve may be a valve that is actively operated by receiving power from the outside by the operation signal. In this embodiment, a valve using a valve material that absorbs electromagnetic wave energy from the outside and operates is employed. The valves 51 and 52 are so-called normally closed valves that shut off the flow of fluid before absorbing electromagnetic energy.

Valve materials include COC (cyclic olefin copolymer), PMMA (polymethylmethacrylate), PC (polycarbonate), PS (polystyrene), POM (polyoxymethylene), PFA (perfluoralkoxy), PVC (polyvinylchloride), PP (polypropylene), PET (polyethylene terephthalate) Thermoplastic resins such as polyetheretherketone (PEEK), polyamide (PA), polysulfone (PSU), and polyvinylidene fluoride (PVDF) may be employed.

As the valve material, a phase change material in a solid state at room temperature may be employed. The phase change material is injected in a molten state between the chambers and solidifies to block the flow of the fluid. The phase change material may be a wax. The wax melts when heated to turn into a liquid state and expands in volume. As the wax, for example, paraffin wax, microcrystalline wax, synthetic wax, natural wax, or the like can be employed. The phase change material may be a gel or a thermoplastic resin. As the gel, polyacrylamide, polyacrylates, polymethacrylates, polyvinylamides, or the like may be employed.

In the phase change material, a plurality of fine exothermic particles absorbing electromagnetic wave energy and generating heat may be dispersed. The micro heating particles have a diameter of 1 nm to 100 μm to allow free passage through the microfluidic passage having a depth of approximately 0.1 mm and a width of 1 mm. The fine exothermic particles have a property of rapidly raising a temperature when the electromagnetic wave energy is supplied by, for example, a laser light or the like, and evenly dispersing the wax. In order to have such properties, the micro heating particles may have a core including a metal component and a hydrophobic surface structure. For example, the micro heating particle may have a molecular structure including a core made of Fe and a plurality of surfactants bound to Fe to surround Fe. The micro heating particles may be stored in a dispersed state in a carrier oil. The carrier oil may also be hydrophobic so that fine exothermic particles having a hydrophobic surface structure can be evenly dispersed. The carrier oil containing fine exothermic particles dispersed in the molten phase-transfer material may be poured and mixed, and the mixture may be injected and coagulated between the chambers to prevent the flow of fluid between the chambers.

The micro heating particles are not limited to the above-mentioned polymer particles, but may also be in the form of quantum dots or magnetic beads. In addition, the micro heating particles may be, for example, a fine metal oxide such as Al ₂ O ₃ , TiO ₂ , Ta ₂ O ₃ , Fe ₂ O ₃ , Fe ₃ O _4, or HfO ₂ . On the other hand, the valve 51, 52 is not necessarily to include the fine heating particles, it may be made of the phase change material only without the fine heating particles. At least part of the platform 1 is transparent so that electromagnetic waves projected from outside the platform 1 can be irradiated to the valves 51 and 52.

Since the reagent is difficult to be stored in the liquid state, a method of making a liquid reagent into a solid reagent is injected into a mold of a liquid phase and lyophilized to prepare a bead having a constant size. Depending on the amount used, a method of injecting an appropriate number of reagent beads into the reagent chamber 30 may be considered. However, it is not always easy to prepare reagent beads of constant size because solid reagents prepared by the lyophilization method may be broken in the process of taking out the mold. In addition, even if the reagents are prepared in the form of beads having the same size by lyophilization, the reagents may be exposed to moisture in the process of injecting the reagents in the bead form into the reagent chamber 30, thereby reducing the performance of the reagents.

As one method for solving the above problems, a method of separating the freezing process and the drying process is considered. Hereinafter, the manufacturing method of the solid phase reagent according to the present embodiment will be described in detail.

First, as shown in FIG. 4A, a mold 300 having a plurality of reagent accommodating parts 301 is prepared. Liquid reagent is injected into the plurality of reagent accommodating portions 301 using an injection means such as a pipette. The liquid phase reagent may be metered in and the volumetric dispersion of the amount may be adjusted to be within 3%. In order to reduce the volume of the reagent to be injected, the concentration of the liquid reagent may be higher than the concentration required to detect the analyte. Dosing reagents means injecting a predetermined amount of reagents from the sample to test the analyte. As an example, a marking may be provided to indicate the quantitative amount in the plurality of receivers to help meter the liquid reagent.

For example, mold 300 may be made of PDMS several millimeters thick. However, the material of the mold 1 is not limited thereto, and any material having chemical and biological stability and flexibility is sufficient. The shapes of the plurality of accommodation portions 301 may all be the same. In addition, as an example, the plurality of accommodation portions 301 may include a plurality of groups having different forms. In this case, different types of reagents can be accommodated in a plurality of groups.

Reagents for blood tests are, for example, serum, aspartate aminotransferase (AST), ALB (Albumin), Alanine Aminotransferese (ALP), alanine aminotransferase (ALT), AMY (Amylase), BUN (Urea Nitrogen), Ca ++ (calcium), CHOL (Total Cholesterol), CK (Creatin Kinase), Cl- (Chloide), CREA (Creatinine), D-BIL (Direct Bilirubin), GGT (Gamma Glutamyl Transferase), GLU (Glucose), HDL (High) -Density Lipoprotein cholesterol (K), Potassisium (K +), Lactate Dehydrogenase (LDH), Low-Density Lipoprotein cholesterol (LDL), Magnesium (Mg), Phosphorus (PHOS), Sodium (Na +), Total Carbon Dioxide (TCO2), T- It may be a reagent for testing Total Bilirubin (BIL), Total Protein (TP), Triglycerides (TRIG), Uric Acid (UA), Albumin (ALB), and Total Protein (TP). Microfluidic devices can be used to test not only blood but also various test substances that can be collected from humans or organisms such as urine.

Fillers may be added to the reagents. The filler is intended to allow the freeze-dried reagent to have a porous structure so that it can be easily dissolved when the diluent mixed with the sample and the diluent is introduced into the reagent chamber 30 later. For example, fillers include bovine serum albumin (BSA), polyethylene glycol (PEG), dextran, mannitol, polyalcohol, myo-inositol, and citric acid ( citric acid), EDTA2Na (ethylene diamine tetra acetic acid disodium salt), and BRIJ-35 (polyoxyethylene glycol dodecyl ether). One or two or more of these fillers may be selected and added depending on the type of reagent.

Surfactants may be added to the reagents. For example, the surfactant may be polyethylene, lauryl ether, octoxynol, polyethylene alkyl alcohol, nonylphenol polyethylene glycol ether; Ethylene oxide (ethylene oxid), ethoxylated tridecyl alcohol, polyoxyethylene nonylphenyl ether phosphate sodium salt, sodium dodecyl sulfate sulfate). One or two or more of these surfactants may be selected and added depending on the type of reagent.

As described above, the mold 300 into which the liquid reagent is injected is placed in a freezer and frozen. Freezing here means to make the water contained in the liquid reagent to the ice state.

As shown in FIG. 4B, the frozen reagent is separated from the mold 300. At this time, since the reagent is in an ice state, it is hard to be broken and maintains a quantitative mass.

The freezing reagent separated from the mold 300 is placed in, for example, the tray 310 shown in FIG. 4C, and the tray 310 is placed in a lyophilizer. Then, a drying program for removing moisture is performed. The drying program may be appropriately set according to the amount or type of reagent. The drying process generally uses sublimation, in which frozen water is directly converted into water vapor, but sublimation is not necessarily used in the whole drying process, but may be used only in part. For sublimation, the pressure of the drying process can be lowered below the triple point of water (6 mbar or 4.6 Torr), but it is not always necessary to maintain a constant pressure. The temperature during the drying process may vary and a method of slowly increasing the temperature after freezing may be used.

As described above, according to the present embodiment, since the freezing process is first performed to freeze the reagent and then separate from the mold 300, the reagent does not break well. Thus, the solid phase reagent after the drying process is completed can maintain a lump state. In addition, a plurality of solid phase reagents can be mass-produced very easily. In addition, since the reagent in a lumped quantity can be injected into the reagent chamber 30, it is very easy to inject the quantitative reagent into the microfluidic device.

The solid phase reagent prepared by the above process is mounted on the reagent chamber 30 provided on the lower plate 11 or the reagent chamber 30 defined by the lower plate 11 and the partition plate 13. Then, the upper plate 12 is bonded to the lower plate 11 or the partition plate 13 to complete the manufacture of the microfluidic device according to an embodiment of the present invention.

Depending on the type of reagents, mixing together and lyophilizing may decrease the activity. Reagents to be separated include reagents for detecting ALP (Alanine Aminotransferese), ALT (alanine aminotransferase), HDL (High-Density Lipoprotein cholesterol), LDL (Low-Density Lipoprotein cholesterol), and the like. Since the presence of a substance and an enzyme present as a substrate in a biochemical reaction simultaneously decreases the titer, this kind of drug should be separated into a reagent containing a substrate and a reagent containing a substance acting as an enzyme. For example, in the case of ALP, the substrate, p-nirophenolphosphate (PNPP), is unstable at pH 10 or higher, and AMP (aminomethanpropanol) and DEA (diethanolamin), which are essential buffers in the reaction system, are high at pH 11-11.5 and are separated and frozen. It should be dry.

One more example, AMY requires sodium chloride (Nacl) in the buffer and substrate. However, sodium chloride is strongly deliquescent, and during freeze-drying, even if it is not lyophilized and freezes, it immediately contains moisture, distorts the properties, and lowers the potency. Therefore, sodium chloride is separately prepared with a buffer.

6 is a schematic configuration diagram of an analyzer using a microfluidic device. Referring to FIG. 6, the rotation driver 510 rotates the microfluidic device 100 to mix a sample, a reagent, and a reagent by centrifugal force. In addition, the rotation driving unit 510 stops the microfluidic device 100 at a predetermined position in order to face the reagent chamber 30 with the detector 520. Although not shown in the drawings, the rotation driver 510 may include a motor drive device capable of controlling the angular position of the microfluidic device 100. For example, the motor drive device may be a step motor or may be a direct current motor. The detector 520 detects optical characteristics such as fluorescence, luminescence, and / or absorbance of a material to be detected, for example. The electromagnetic wave generator 530 is for operating the valves 51 and 52 and, for example, irradiates laser light.

Sample analysis can be carried out in the following manner. A diluent such as a buffer solution or distilled water is injected into the dilution chamber 20 of the microfluidic device 100, and a sample such as blood collected from a subject or serum separated therefrom is injected into the sample chamber 10.

Then, the microfluidic device 100 is mounted to the analyzer as shown in FIG. 6. In the case of the chip type microfluidic device 100 that cannot be directly mounted on the rotary drive unit 510, the microfluidic device 100 is mounted on the adapter 540, and the adapter 540 is mounted on the rotary drive unit 510. I can attach it. At this time, the fluid should flow from the sample chamber 10 toward the reagent chamber 30 so that the sample chamber 10 is located on the inside and the reagent chamber 30 is located on the outside based on the center of rotation of the adapter 540. The microfluidic device 100 is mounted. The rotation driver 510 rotates the microfluidic device 100 to face the valve 51 with the electromagnetic wave generator 530. When the electromagnetic wave is irradiated to the valve 51, the passage between the sample chamber 10 and the dilution chamber 20, as shown in Figure 5 while melting the valve material constituting the valve 51 by the energy Open. The sample is introduced into the dilution chamber 20 by centrifugal force. The rotary drive unit 510 shakes the microfluidic device 100 from side to side to form a sample diluent by mixing the sample and the diluent. Then, the electromagnetic wave is also irradiated to the valve 52 to open the passage between the dilution chamber 20 and the reagent chamber 30, and supply the sample diluent to the reagent chamber 30. Then, the solid reagent contained in the reagent cartridge 200 is mixed with the sample diluent to melt. In order to melt and mix the solid reagent with the sample diluent, the rotation driver 510 may shake the microfluidic device 100 from side to side several times. As a result, a reagent mixture is formed in the reagent chamber 30.

Then, the reagent chamber 30 is faced with the detector 520 to detect whether the substance to be detected is present in the reagent mixture in the reagent chamber 30 and the amount thereof.

7 shows a second embodiment of the microfluidic device according to the present invention. The microfluidic device 102 shown in FIG. 7 is in the form of a disc that can be mounted directly to the rotary drive unit 510 of the analyzer. Although only a part thereof is shown in FIG. 7, the platform 1 is in the form of a circular disk. The platform 1 can be a two-plate structure as shown in FIG. 2 or a three-plate structure as shown in FIG. 3.

The platform 1 is provided with a sample chamber 10, a dilution chamber 20, and a reagent chamber 30. The reagent chamber 30 is located at a position far from the center of rotation of the platform 1 relative to the sample chamber 10 and the dilution chamber 20. The sample chamber 10 is connected to the dilution chamber 20, and the dilution chamber 20 is connected to the reagent chamber 30. Valves 51 and 52 are provided between the sample chamber 10 and the dilution chamber 20 and between the dilution chamber 20 and the reagent chamber 30, respectively. The reagent chamber 30 contains a solid reagent.

The microfluidic device 103 illustrated in FIG. 8 is a variation of the microfluidic device 102 illustrated in FIG. 7. The sample chamber 10 and the dilution chamber 20 are connected to the reagent chamber 30. Valves 51 and 52 are provided between the sample chamber 10 and the reagent chamber 30 and between the dilution chamber 20 and the reagent chamber 30, respectively.

Sample analysis can be carried out in the following manner. The dilution liquid 20 such as a buffer solution or distilled water is injected into the dilution chamber 20 of the microfluidic device 102 or 103. Into the sample chamber 10, a sample such as blood collected from a subject or serum separated therefrom is injected.

Then, the microfluidic device 102 or 103 is mounted to the rotary drive unit 510 of the analyzer as shown in FIG. The rotation drive unit 110 rotates the microfluidic device 102 or 103.

Next, the rotary drive unit 510 faces the valve 51, 52 and the electromagnetic wave generator 530, respectively. When electromagnetic waves are irradiated to the valves 51 and 52, the valve substances constituting the valves 51 and 52 are melted by the energy. When the microfluidic device 102 or 103 is rotated, the sample and the diluent are introduced into the reagent chamber 30 by centrifugal force. The solid reagent contained in the reagent chamber 30 is mixed with the sample diluent mixed with the sample and diluted to melt. In order to dissolve the solid phase reagent, the rotation driver 510 may perform an operation of shaking the microfluidic device 103 or 103 several times from side to side. Then, the reagent chamber 30 is faced with the detector 520 to detect whether the substance to be detected is present in the reagent mixture in the reagent chamber 30 and the amount thereof.

9 shows a third embodiment of the microfluidic device according to the present invention. Referring to Figure 9, the microfluidic device 104 of the present embodiment is in the form of a disk that can be mounted directly on the rotary drive unit 510 of the analyzer, and has a centrifugation unit 70 for separating the supernatant from the sample. For example, when whole blood is injected as a sample, the serum and sediment are separated by the centrifugation unit 70. The platform 1 is in the form of a circular disk. The platform 1 can be a two-plate structure as shown in FIG. 2 or a three-plate structure as shown in FIG. 3.

The side close to the center of the platform 1 is called the inside, and the side far from the center is called the outside. The sample chamber 10 is disposed at the innermost side of the platform 1. The centrifugal separation unit 70 includes a centrifugal separator 71 positioned outside the sample chamber 10 and a sediment collector 72 positioned at the end of the centrifugal separator 71. Substantially the sample chamber 10 may also be part of the centrifuge unit 70. When the sample is centrifuged, the supernatant is collected in the sample chamber 10 and the centrifuge 71, and the large sediment is moved to the sediment collection unit 72.

Dilution liquid is accommodated in the dilution chamber 20. The centrifugal separator 71 and the dilution chamber 20 are connected to the mixing chamber 80. Valves 51 and 52 are respectively provided between the centrifugal separator 71 and the mixing chamber 80 and between the dilution chamber 20 and the mixing chamber 80.

In the outermost part of the platform 1, a plurality of reagent chambers 30 are provided. The mixing chamber 80 is connected to the plurality of reagent chambers 30 by the channel 45. The channel 45 is provided with a valve 55. The valve 55 may be the same valve as the valves 51 and 52. Solid reagents are accommodated in the plurality of reagent chambers 30.

Sample analysis can be carried out in the following manner. A diluent of a liquid such as a buffer solution or distilled water is injected into the dilution chamber 20 of the microfluidic device 105. A sample such as blood collected from the subject is injected into the sample chamber 10.

Then, the microfluidic device 105 is mounted on the rotary drive unit 510 of the analyzer as shown in FIG. The rotation driver 110 rotates the microfluidic device 104. Then, in the sample contained in the sample chamber 10, the supernatant is collected in the sample chamber 10 and the centrifugal separator 71 by centrifugal force, and the material having a large mass is collected in the sediment collector 72.

Next, the rotary drive unit 510 faces the valve 51, 52 and the electromagnetic wave generator 530, respectively. When electromagnetic waves are irradiated to the valves 51 and 52, the valve substances constituting the valves 51 and 52 are melted by the energy. When the microfluidic device 104 is rotated, the sample and the diluent are introduced into the mixing chamber 80 by centrifugal force. In the mixing chamber 80, a sample diluent in which the sample and the diluent are mixed is formed. In order to mix the sample and the diluent, the rotary driver 510 may shake the microfluidic device 104 from side to side several times.

Next, the rotation driver 510 faces the valve 55 with the electromagnetic wave generator 530. When electromagnetic waves are irradiated to the valve 55, the channel 45 is opened while the valve material constituting the valve 55 is melted by the energy. When the microfluidic device 104 is rotated, the sample diluent is introduced into the reagent chamber 30 through the channel 45 by centrifugal force. The solid reagent is mixed with the sample diluent and dissolved. In order to dissolve the solid phase reagent, the rotation driver 510 may shake the microfluidic device 104 from side to side several times.

Then, the reagent chamber 30 is faced with the detector 520 to detect whether the substance to be detected is present in the reagent mixture in the reagent chamber 30 and the amount thereof.

Using the microfluidic device 103 shown in FIG. 9, a detection process requiring a two-step reaction, for example, a process of detecting HDL from a sample will be described. In this case, as shown in FIG. 10, the solid reagent 1 is accommodated in the first reagent chamber 33. In the second reagent chamber 34, the solid reagent 1 and the reagent 2 are accommodated. The composition of Reagent 1 and Reagent 2 is as follows.

<Reagent 1>

Piperazine-1,4-bis (2-ethanesulfonic acid): 30 MMOL / L

4-AAP (4-aminoantipyrine): 0.9 MMOL / L

POD (peroxidese): 240 U / L

ASOD (ascobic oxidase): 2700 U / L

anti hunam b-lipoprotein antibody

<Reagent 2>

PIPES (Piperazine-1,4-bis (2-ethanesulfonic acid)): 30 MMOL / L

Cholesterol esterase (CHE): 4000 U / L

Cholesterol oxidase (COD): 20000 U / L

H-DASO (N- (2-hydrox-3sulfopropyl) -3.5-dimethoxyaniline): 0.8 MMOL / L

In the first reagent chamber 33, the reagent 1 and the sample diluent are mixed and held at about 37 ° C for 5 minutes. At this time, HDL LDL, very low density lipoprotein (VLDL), chylomicron becomes soluble HDL, soluble HDL is decomposed into cholesterol and hydrogen peroxide. Hydrogen peroxide breaks down into water and oxygen.

In the second reagent chamber 34, the reagent 1, the reagent 2, and the sample dilution liquid are mixed and maintained at about 37 ° C for 5 minutes. At this time, HDL LDL, VLDL, and chylomicron become soluble HDL by the enzymatic reaction of Reagent 1, and soluble HDL is decomposed into cholestenone and hydrogen peroxide. Hydrogen peroxide breaks down into water and oxygen. The remaining HDL forms a pigment by enzymatic reaction with reagent 2. The absorbance is measured by irradiating light to the first and second reagent chambers 33 and 34 using the detector 520.

The presence or absence of HDL may be measured from the absorbance measurement results of the two steps.

11 shows a fourth embodiment of the microfluidic device according to the present invention. 12A and 12B are cross-sectional views of a fourth embodiment of the microfluidic device according to the present invention shown in FIG. In the present embodiment, the microfluidic device 105 is coupled to a container 90 containing a diluent on the platform 1, and the container 90 is connected to the dilution chamber 20 by a channel 43. There is a difference from the microfluidic device 104 shown. The channel 53 may be provided with a valve 53. The valve 53 may be the same valve as the valves 51 and 52 described above. Of course, since the flow of the diluent may be controlled by the lid 95 to be described later, the valve 53 may not be provided in the channel 43.

Referring to FIGS. 11, 12A, and 12B, the platform 1 has a shape in which the upper plate 12 and the lower plate 11 are bonded to each other. The container 90 has an accommodation space 91 for accommodating the diluent. The container 90 can be produced, for example, by injection molding a thermoplastic resin and is fixed to the platform 1. The accommodation space 91 is sealed by a lid 95. The container 90 is turned upside down to inject a diluent into the accommodating space 91 to fill it, and the lid 95 is adhered to the opening 93 of the container 90 to prevent the dilution from flowing out. Inside the container 90, a fluid pouch may be provided that receives the diluent and is rupturably sealed.

The lid 95 is an example of a control means for controlling the flow of the diluent from the container 90 through the channel 43. The lid 95 prevents an arbitrary outflow of the diluent contained in the accommodation space 91, and provides energy, supplied from the outside, For example, it is broken or melted by the energy of electromagnetic waves such as laser light.

For example, the lead 95 may include a thin metal film and an electromagnetic wave absorbing layer laminated thereon. The electromagnetic wave absorbing layer is formed by applying an electromagnetic wave absorbing material. Due to the electromagnetic wave absorbing layer, the lead 95 absorbs and bursts or melts electromagnetic waves projected from the outside. As long as the thin film is capable of being ruptured or melted by electromagnetic wave irradiation, other materials such as a polymer may be employed as well as a metal. At least a portion of the container 90 is transparent so that electromagnetic waves projected from outside the container 90 can pass through the container 90 and irradiate the lid 95.

When the microfluidic device 105 is mounted on the rotary drive unit 510 of the analyzer shown in FIG. 6, and the electromagnetic wave is irradiated to the lead 95 for a predetermined time using the electromagnetic wave generator 530, as shown in FIG. 12B. The lid 95 ruptures or melts.

Next, when electromagnetic waves are irradiated to the valve 53 using the electromagnetic wave generator 530, the valve material constituting the valve 53 is melted to open the channel 43. The diluent that was accommodated in the accommodation space 91 passes through the channel 43 to the dilution chamber 20 to be received. Thereafter, the analysis process is the same as the analysis process using the microfluidic device 103 of FIG.

Although embodiments according to the present invention have been described above, these are merely exemplary, and it will be understood by those skilled in the art that various modifications and equivalent other embodiments are possible. Therefore, the protection scope of the present invention should be defined by the appended claims.

1 is a plan view showing a first embodiment of a microfluidic device according to the present invention.

2 is a view showing an example of a microfluidic device having a two-plate structure.

3 is a view showing an example of a microfluidic device having a three-plate structure.

4A to 4C are diagrams illustrating one embodiment of a method for preparing a solid phase reagent according to the present invention.

5 is a cross-sectional view showing the channel is opened by the valve.

6 is a schematic structural diagram of an example of an analyzer using a microfluidic device.

7 is a plan view showing an embodiment having a disk type platform as a second embodiment of the microfluidic device according to the present invention;

8 is a plan view showing one modification of the second embodiment of the microfluidic device shown in FIG.

Figure 9 is a plan view showing an embodiment having a centrifugal separation unit as a third embodiment of the microfluidic device according to the present invention.

10 is a view for explaining the detection process by the two-step reaction using the microfluidic device shown in FIG.

11 is a plan view showing an embodiment having a container for supplying a diluent as a fourth embodiment of the microfluidic device according to the present invention;

12A and 12B are cross-sectional views of FIG. 11;

<Description of the symbols for the main parts of the drawings>

1 ...... Platform 11 ...... Bottom

12 ...... Top 13 ... Compartment

10 ...... sample chamber 20 ...... dilution chamber

30 ..... Reagent chamber 43, 45 ...... channel

51-53, 55 ...... Valve 70 ... Centrifuge Unit

71 ...... centrifuge 72 ...... Sedimentary collection part

80 ...... Mixed chamber 90 ...... Container

95 ...... Lead 100-105 ...... Microfluidic device

510 ...... rotation drive 520 ...... detector

530 ...... electromagnetic generator 540 ...... adapter

Claims (20)

Preparing a mold in which a plurality of reagent receivers are formed; Injecting a liquid reagent into the plurality of reagent receivers; Freezing the liquid reagent; Separating the frozen reagent from the mold; Removing the water of the frozen reagent and drying the solid reagent. The method of claim 1, wherein the drying step, Method for producing a solid phase reagent comprising the step of subliming the water contained in the frozen reagent. The method of claim 1, The liquid reagent is concentrated to a concentration higher than the concentration used in the test method for producing a solid reagent in the plurality of reagent receiving portion. The method of claim 1, Solid reagents containing at least two or more liquid reagents in the plurality of reagent receiving portion. The method of claim 1, The method of manufacturing a solid reagent of the plurality of accommodating portion is at least two kinds. The method of claim 1, The mold is a method of manufacturing a solid reagent of a flexible material. The method of claim 1, The reagent is serum, aspartate aminotransferase (AST), ALB (Albumin), ALP (Alanine Phosphatase), ALT (alanine aminotransferase), AMY (Amylase), BUN (Urea Nitrogen), Ca ++ (calcium), CHOL ( Total Cholesterol), CK (Creatin Kinase), Cl- (Chloide), CREA (Creatinine), D-BIL (Direct Bilirubin), GGT (Gamma Glutamyl Transferase), GLU (Glucose), HDL (High-Density Lipoprotein Cholesterol), Potassium (K +), Lactate Dehydrogenase (LDH), Low-Density Lipoprotein Cholesterol (LDL), Magnesium (Mg), Phosphorus (PHOS), Sodium (Na +), Total Carbon Dioxide (TCO2), Total Bilirubin (T-BIL), TP (Total Protein), TRIG (Triglycerides), UA (Uric Acid), ALB (Albumin), TP (Total Protein) method for producing a solid reagent selected from the reagents for testing. The method of claim 1, Method for producing a solid reagent with a filler is added to the liquid reagent. The method of claim 8, The filler is BSA (bovine serum albumin), PEG (polyethylene glycol), dextran (dextran), mannitol, polyalcohol, myo-inositol, citric acid (citric acid) , EDTA2Na (ethylene diamine tetra acetic acid disodium salt), BRIJ-35 (polyoxyethylene glycol dodecyl ether) method for producing a solid phase reagent. The method of claim 1, The liquid reagent is a method for producing a solid phase reagent is added to the surfactant. The method of claim 10, The surfactant may be polyethylene (polyoxyethylene), lauryl ether (lauryl ether), octoxynol, polyethylene alkyl alcohol, nonylphenol polyethylene glycol ether; Ethylene oxide (nonylphenol polyethylene glycol ether; ethylene oxid), ethoxylated tridecyl alcohol, polyoxyethylene nonylphenyl ether phosphate sodium salt, sodium dodecyl sulfate sulfate). A sample chamber in which a sample to be inspected is accommodated; A dilution chamber in which a diluent is accommodated; A reagent chamber containing a solid phase reagent; And a channel connecting the chambers. The method of claim 12, And a valve for controlling the flow of fluid through the channel. The method of claim 13, The valve is a microfluidic device using a material that is opened by electromagnetic energy as a valve material. The method of claim 14, The valve material is a microfluidic device selected from a phase change material or a thermoplastic resin whose phase is changed by energy. The method of claim 15, The valve material is a microfluidic device that is dispersed in the phase change material and includes micro heating particles absorbing energy of electromagnetic waves to generate heat. A substrate provided with a plurality of chambers; A solid reagent contained in at least one of the plurality of chambers, The solid phase reagent is a microfluidic device that is a quantitative reagent. The method of claim 17, A sample chamber in which a sample to be inspected is accommodated; A dilution chamber in which a diluent is accommodated; And a plurality of reagent chambers in which the solid phase reagent is accommodated. The method of claim 18, Two or more kinds of the solid reagent are accommodated in the plurality of reagent chambers, The solid reagent is a microfluidic device is different in shape depending on the type. The method according to any one of claims 17 to 19, A channel connecting the plurality of chambers; And a valve installed in the channel to control the flow of fluid through the channel, the valve blocking the channel in a solidified state and melting by electromagnetic wave energy to open the channel.

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